Tumor-specific P450 protein

ABSTRACT

The discovery that CYP1B1 protein is detectable in a wide range of human cancers of different histogenetic types, but is not detectable in non-cancerous tissues, gives rise to diagnostic methods for detecting tumors based on this protein as a marker, and to the possibility of tumor therapies involving the protein. A diagnostic method may include the steps of (a) obtaining from a patient a tissue sample to be tested for the presence of cancer cells; (b) producing a prepared sample in a sample preparation process; (c) contacting the prepared sample with an antibody that reacts with human CYP1B1 protein; and (d) detecting binding of the antibody to CYP1B1 protein in the prepared sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/874,166, filed on Jun. 4, 2001, which is a divisional of U.S.application Ser. No. 09/043,814, filed on Jan. 22, 1999, issued as U.S.Pat. No. 6,242,203 on Jun. 5, 2001, which is a continuation under 35 USC§371 of PCT Application Number PCT/GB96/02368, filed on Sep. 25, 1996,which claims priority from Great Britain Application Number 9519490.8,filed on Sep. 25, 1995, all of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

[0002] This invention relates to tumor diagnosis and therapy, and tomaterials and methods for use therein.

[0003] More particularly, the invention is based on the identificationof a cytochrome P450 form, specifically CYP1B1, in a wide range oftumors, with a high frequency of expression in each type, and proposesthe use of this enzyme as a tumor marker, and as the basis of aselective therapeutic approach involving the design of drugs, e.g.,which are activated to a cytotoxic form by the action of CYP1B1.

BACKGROUND

[0004] The major goal of cancer chemotherapy is the development ofanti-cancer drugs that are effective in a wide range of cancers andproduce no toxic effects in normal tissues. The target of such drugsshould be expressed only in tumor cells and not normal cells. However,to date, no such tumor specific target, general to all types of cancers,has been identified.

[0005] The cytochromes P450 are a multi-gene family of constitutive andinducible enzymes, which have a central role in the oxidative metabolicactivation and detoxification of both a wide range of xenobiotics (2-4)and several groups of endogenous compounds active in cell regulation andcell signaling including arachidonic acid (5), steroid hormones (6) andfatty acids (7). The major families of P450 involved in xenobioticmetabolism each consist of several individual forms with differentregulatory mechanisms and substrate specificities (2). The majority ofP450s are primarily expressed in liver (2) although individual P450forms are also expressed in specific extra-hepatic tissues (8) includingsmall intestine, kidney and lung.

[0006] The human CYP1 gene family (individual P450 forms are identifiedby the prefix CYP in accordance with the current P450 nomenclature (3)),which is one of the major P450 families involved in the metabolism ofxenobiotics, is now known to consist of three individual formsclassified into two sub-families. The CYP1A subfamily contains twohighly homologous and well characterized but distinct members, CYP1A1(9) and CYP1A2 (10). CYP1A1 is an inducible P450 expressed primarily inextrahepatic tissues (11) while CYP1A2 is a major form of P450 that isconstitutively expressed in liver (12). Recently a second human CYP1subfamily has been identified which to date contains one member, CYP1B1(1). This P450 is dioxin-inducible, and sequence analysis of CYP1B1shows 40% homology with both CYP1A1 and CYP1A2. Although CYP1B1 isassigned to the CYP1 family on the basis of its sequence, it appears tobe structurally distinct from both CYP1A1 and CYP1A2.

[0007] Several forms of P450 are considered to have an important role intumor development since they can metabolize many potential carcinogensand mutagens (13). Moreover, P450 activity may influence the response ofestablished tumors to anti-cancer drugs; several cancer chemotherapeuticagents can be either activated or detoxified by this enzyme system (14).The presence of individual forms of P450 had previously beeninvestigated in different types of cancer including breast cancer (15),lung cancer (16), colon cancer (17) and head and neck cancer (18) todetermine if intra-tumor metabolism of anti-cancer agents by P450 couldoccur and thus influence the response of tumors to these agents. Thesestudies have generally shown that the level of the P450 formsinvestigated is significantly reduced or absent in tumors when comparedwith the adjacent normal tissue in which the tumors have developed.However, our recent studies of several different types of cancer (19)including breast cancer, esophageal cancer and soft tissue sarcomas haveshown that there may be tumor-specific expression of a CYP1 form ofP450.

[0008] Although CYP1B1 mRNA had previously been identified by Northernblotting in several normal human tissues (1), the presence of CYP1B1protein itself had not been demonstrated.

SUMMARY

[0009] The present invention is based on the discovery that CYP1B1 is atumor-specific form of P450, present in a wide range of malignant tumorsand not detected in normal tissues.

[0010] Accordingly, a first aspect of the present invention provides amethod for the identification of tumor cells, which method comprises theuse of a recognition agent, for example an antibody, recognizing CYP1B1protein to contact a sample of tissues, cells, blood or body product, orsamples derived therefrom, and screening for a positive response. Thepositive response may, for example, be indicated by an agglutinationreaction or by a visualizable change such as a color change orfluorescence, e.g. immunostaining, or by a quantitative method such asin the use of radio-immunological methods or enzyme-linked antibodymethods.

[0011] The method therefore typically includes the steps of (a)obtaining from a patient a tissue sample to be tested for the presenceof cancer cells; (b) producing a prepared sample in a sample preparationprocess; (c) contacting the prepared sample with a recognition agent,such as an antibody, that reacts with human CYP1B1 protein; and (d)detecting binding of the recognition agent to CYP1B1 protein, ifpresent, in the prepared sample. The human tissue sample can be from forexample the bladder, brain, breast, colon, connective tissue, kidney,lung, lymph node, esophagus, ovary, skin, stomach, testis, and uterus.

[0012] A preferred sample preparation process includes tissue fixationand production of a thin section. The thin section can then be subjectedto immunohistochemical analysis to detect binding of the recognitionagent to CYP1B1 protein. Preferably, the immunohistochemical analysisincludes a conjugated enzyme labeling technique. A preferred thinsection preparation method includes formalin fixation and wax embedding.Alternative sample preparation processes include tissue homogenization,and preferably, microsome isolation. When sample preparation includestissue homogenization, a preferred method for detecting binding of theantibody to CYP1B1 protein is Western blot analysis. Alternatively, animmunoassay can be used to detect binding of the antibody to CYP1B1protein. Examples of immunoassays are antibody capture assays,two-antibody sandwich assays, and antigen capture assays. Preferably,the immunoassay is a solid support-based immunoassay. When Western blotanalysis or an immunoassay is used, preferably it includes a conjugatedenzyme labeling technique.

[0013] Although the recognition agent will conveniently be an antibody,other recognition agents are known or may become available, and can beused in the present invention. For example, antigen binding domainfragments of antibodies, such as Fab fragments, can be used. Also,so-called RNA aptomers may be used (36, 37). Therefore, unless thecontext specifically indicates otherwise, the term “antibody” as usedherein is intended to include other recognition agents. Where antibodiesare used, they may be polyclonal or monoclonal. Optionally, the antibodycan produced by a method so that it recognizes a preselected epitope ofsaid CYP1B1 protein.

[0014] A second aspect of the invention lies in the presence of CYP1B1protein selectively in tumors, e.g. in kidney tumors and not normalrenal tissue, combined with the absence of CYP1B1 protein expression innormal liver, which provides a mechanism for the selective targeting ofanti-cancer drugs based on CYP1B1 metabolism in tumors. Drugs can bedesigned for, or screened for, specific metabolism by CYP1B1 in tumorswhereby this metabolism converts a non-toxic moiety into a toxic one,which kills or inhibits the tumor or makes it more susceptible to otheragents.

[0015] A third aspect of the invention provides for the targeting ofcytotoxic drugs or other therapeutic agents, or the targeting of imagingagents, by virtue of their recognition of CYP1B1 epitopes on the surfaceof a tumor cell, whether as part of the complete CYP1B1 protein itselfor in some degraded form such as in the presentation on the surface of acell bound to a MHC protein.

[0016] Another aspect of the invention provides stimulation of theimmune system of cancer patients, for example by activating cytotoxic orhelper T-cells which recognize CYP1B1 epitopes so as to implement acell-mediated or humoral immune response against the tumor. Theactivation of the immune system can be achieved by immunization withCYP1B1 sequences.

[0017] Because the expression of CYP1B1 is very common in tumors of manydifferent types, it is likely that this enzyme performs an essentialfunction for the tumor cells, for example by inactivating endogenousanti-tumor compounds such as 2-methoxyestradiol.

[0018] Consequently, another aspect of the invention is the reduction ofCYP1B1 levels in tumor cells, for example, by the use of suicideinhibitors or by using antisense RNA methods to decrease the synthesisof the protein. Down-regulation of the CYP1B1 promoter could alsoachieve the reduction in CYP1B1 levels.

DESCRIPTION OF DRAWINGS

[0019]FIG. 1 shows SDS-PAGE and immunoblotting procedures using ananti-CYP1B1 antibody to look for CYP1B1 protein in normal and tumorouskidney tissue and in normal liver tissue.

[0020]FIG. 2 shows SDS-PAGE and immunoblotting to detect CYP1B1 proteinin breast tumor and in normal liver tissue.

[0021]FIG. 3 shows an immunoblot of CYP1B1 in different types of tumorsand normal tissues.

[0022]FIG. 4 shows CYP1B1 and β-actin mRNA in normal (A and B) andcorresponding tumor (C and D) samples which have been detected byRT-PCR.

DETAILED DESCRIPTION

[0023] Expression of CYP1B1 was investigated in different types ofcancers that had developed in a broad range of different anatomicalsites (bladder, breast, colon, kidney, lung, esophagus, ovary, skin,stomach, uterus, bone and connective tissue, lymph node, brain andtestis). Primary malignant tumors of these tissues constitute differenthistogenetic types (carcinomas, lymphomas, sarcomas, neuro-epithelialtumors and germ cell tumors) each with a different biological behavior.These tumors also represent a range of both common and less common typesof cancer. The presence of CYP1B1 was also investigated in a widevariety of normal tissues.

[0024] Immunohistochemistry for CYP1B1 showed that in all the differenttypes of tumor there was strong immunoreactivity for CYP1B1. CYP1B1immunoreactivity was localized specifically to tumor cells. Non-tumorcells including stromal cells, inflammatory cells, and endothelial cellspresent in the sections of tumor showed no immunoreactivity for CYP1B1.There was no significant intra-tumor heterogeneity of CYP1B1immunoreactivity and only in five out of 133 tumors was CYP1B1 notdetected. There was no immunoreactivity for CYP1B1 in any of the normaltissues studied which included liver, kidney, small intestine and lung.

[0025] The absence or low level of individual forms of P450 in moststudies of human cancer (15-18), combined with extrapolation fromstudies of rodent hepatic carcinogenesis (25), had led to the generalbelief that tumor cells do not significantly express P450. However, wehave now shown that CYP1B1 is expressed in a wide variety of malignanttumors of different histogenetic types and is not present in normaltissues, indicating that this P450 is a tumor specific form of P450.Tumors are composed of a variable proportion of tumor cells andnon-tumor cells. To identify that a protein is tumor specific, it isimportant to demonstrate that the protein is localized only to tumorcells. Immunohistochemistry allows the direct visualization of tumorcells and has the spatial resolution to separate tumor cells fromnon-tumor cells. Furthermore, it is important to show there has been nodifferential degradation of proteins in normal tissue samples comparedwith tumor samples, and immunoblotting for β-actin (as a positivecontrol protein) showed it to be present in every normal and tumorsample indicating there was no protein degradation. In addition,Coomassie blue staining of the polyacrylamide gels showed no evidence ofprotein degradation. Moreover, immunohistochemistry of the tumor samplesprovides its own internal control as sections of tumor contain non-tumorcells.

[0026] The presence of CYP1B1 in many types of tumor suggests that thisP450 may have a crucial endogenous function in tumor cells and CYP1B1may contribute to drug resistance that is observed in many types oftumor. CYP1B1 is also likely to be important in tumor development andprogression. Its identification in a diverse range of cancers ofdifferent histogenetic types and its absence from normal tissues appearsto make CYP1B1 one of the common changes of a gene product in malignancy(26).

[0027] A previous investigation (1) found mRNA in normal tissues. Insome tumors, increased (2-4×) mRNA was found compared with normal. Thismight be due to increased transcription mediated by hypoxia induciblefactor. This is a novel heterodimeric transcription factor which isinduced by hypoxia, and the stimulus in this case may be the hypoxicmicro-environment that can exist in tumors, and this factor can have asone of its components the Ah receptor nuclear translocator (27).However, regulation of other forms of P450 is complex (2, 28) and theregulation of CYP1B1 in tumors is likely to be complex also, withmultiple mechanisms including transcriptional and post-transcriptionalfactors involved.

[0028] The tumor-specific expression of CYP1B1 has importantconsequences for both the diagnosis and treatment of cancer. Newdiagnostic procedures based on the presence of CYP1B1 in cancer cellscan be developed, while the expression of CYP1B1 in tumor cells providesa molecular target for the development of new anti-cancer drugs that areselectively activated by CYP1B1 in tumor cells. Since CYP1B1 is found ina wide range of tumors it would be expected that such drugs would beeffective in treating many different types of cancer. An importantfeature is that it would be anticipated these drugs would not beassociated with the systemic toxicity that limits the use of currentanti-cancer drugs as CYP1B1 is not present in normal tissues especiallyliver, small intestine and kidney that are the main tissues involved indrug metabolism. Thus, a major problem to targeting anticancer drugs attumors based on their selective activation by P450 has been markedhepatic P450 metabolism of drugs resulting in decreased bioavailabilityand/or undue toxicity. The absence of CYP1B1 protein in liver overcomesthis problem.

[0029] As regards tumor diagnosis, numerous methods for using antibodiesto detect a specific protein, including CYP1B1, in a biological sampleare known and can be used in the present invention. Any of the variousantibody methods can be used alone in practicing the present invention.If desired, two or more methods can be used to complement one another.

[0030] A preferred method for use in the present invention isimmunohistochemical analysis. Immunohistochemical analysisadvantageously avoids a dilution effect when relatively few cancer cellsare in the midst of normal cells. An early step in immunohistochemicalanalysis is tissue fixation, which preserves proteins in place withincells. This prevents substantial mixing of proteins from differentcells. As a result, surrounding normal cells do not diminish thedetectability of CYP1B1-containing cancer cells. This is in contrast toassay methods that involve tissue homogenization. Upon tissuehomogenization, CYP1B1 protein from cancer cells is mixed with proteinsfrom any surrounding normal cells present in the tissue sample. Theconcentration of CYP1B1 protein is thus reduced in the prepared sample,and it can fall below detectable limits. Immunohistochemical analysishas at least three other advantages. First, it requires less tissue thanis required by alternative methods such as Western blot analysis orimmunoassay. Second, it provides information on the intracellularlocalization and distribution of immunoreactive material. Third,information on cell morphology can be obtained from the same thinsection used to test for the presence of CYP1B1 protein. Preferably,when immunohistochemical analysis is employed in the practice of thisinvention, several thin sections from each tissue sample are preparedand analyzed. This increases the chances of finding small tumors.

[0031] Another preferred antibody method for use in the presentinvention is Western blot analysis, i.e., sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed byimmunoblotting. Sample preparation for Western blot analysis includestissue homogenization, and optionally isolation of microsomes. Westernblot analysis has the advantage of detecting immunoreactivity onproteins that have been separated with high resolution, according to(apparent) molecular weight.

[0032] Immunoassays such as antibody capture assays, two-antibodysandwich assays, and antigen capture assays can also be used in thepresent invention. Sample preparation for immunoassays includes tissuehomogenization, and optionally isolation of microsomes. Immunoassayshave the advantage of enabling large numbers of samples to be testedrelatively quickly, and they offer quantitative precision.

[0033] Principles and practice of immunohistochemistry, Western blotanalysis, and immunoassays are well known. One of ordinary skill in theart can select suitable protocols and carry out immunohistochemicalanalysis, Western blot analysis, or an immunoassay, in the practice ofthe present invention (30).

[0034] Experimental Details

[0035] 1. Preparation of Antibodies

[0036] As already noted, the diagnostic aspects of the present inventioncan conveniently use an antibody that recognizes human CYP1B1. Antibodyspecificity for CYP1B1 protein is preferable, but not required.Preferably, any non-CYP1B1 protein recognized by the antibody is readilydistinguished from CYP1B1, e.g., according to apparent molecular weighton a Western blot. With selection of an appropriate assay-protocol,which is within ordinary skill in the art, the invention can bepracticed with a polyclonal antibody or a monoclonal antibody.

[0037] A polyclonal antibody or monoclonal antibody suitable for use inthe present invention can be obtained according to conventionalprocedures (30). Preparation of antibodies that react with CYP1B1protein is known (31). Procedures for obtaining antibodies that reactwith human CYP1B1 protein can be carried out using a preparation ofnon-human CYP1B1 protein, e.g., murine CYP1B1 protein. A CYP1B1 proteinpreparation suitable for eliciting antibodies useful in the presentinvention can be obtained according to various procedures, includingthose described (31).

[0038] Antibodies useful in the present invention can be obtained byimmunizing an animal with a preparation containing intact CYP1B1protein. Alternatively, useful antibodies can be obtained by immunizingan animal with a polypeptide or oligopeptide corresponding to one ormore epitopes on the CYP1B1 protein.

[0039] Preparation

[0040] To prepare an antibody according to the latter approach, two15-mer peptides corresponding to epitopes on the human CYP1B1 proteinwere synthesized. Each corresponded to a different putative surface loopregion of the CYP1B1 enzyme. The first peptide (designated 217A)consisted of 14 amino acids, i.e., ESLRPGAAPR DMMD (SEQ ID NO: 1).Peptide 217A represented amino acid positions 312-325 of the deducedamino acid sequence. A carboxy terminal cysteine was included for use ina conjugation reaction. The second peptide (designated 218A) consistedof 14 amino acids, i.e., EKKAAGDSHG GGAR (SEQ ID NO: 2). Peptide 218Arepresented positions 332-345 of the deduced amino acid sequence. Acarboxy terminal cysteine was added for use in a conjugation reaction.Each of these peptides was conjugated directly to KLH.

[0041] Male New Zealand rabbits were immunized at several anatomicalsites using 100 μg of the 217A peptide conjugate or the 218A peptideconjugate. The conjugates were dissolved in 300 μl of PBS mixed with 300μl of Freund 's Complete Adjuvant. Three weeks after the initialimmunization, the rabbits were boosted with 50 μg of one of each of theconjugates (contained in 300 μl PBS mixed with 300 μl of Freund 'sIncomplete Adjuvant, injected in several sites). One week later (fourweeks after the initial injection), the rabbits were boosted again,using the same protocol. One week after this second boost, the firstserum sample was collected. Rabbits were subsequently boosted, and serumsamples were collected, weekly. Serum samples were screened foranti-CYP1B1 titer and specificity by Western blotting against a humanCYP1B1-maltose binding fusion protein expressed in E. coli, and humanCYP1B1 protein expressed in COS-1 cells.

[0042] Anti-CYP1B1 IgG was purified by immunoafftinity chromatography.The chromatography was carried out using the appropriate CYP1B1 peptidelinked directly to a commercial N-hydroxysuccinamide ester of aderivatized, cross-linked agarose gel bead support (AffiGel 10; Biorad,Richmond, Calif.). Conjugation and chromatography were performedaccording to the vendor 's recommended protocols.

[0043] 2. Detection of CYP1B1 Protein and its mRNA

[0044] In general in the experiments that follow, samples of normaltissue were obtained from tissue specimens that were removed frompatients undergoing surgery for malignant disease. Normal liver, stomachand small intestine were also obtained from organ transplant donors. Allthe tissue samples were processed immediately after excision to preventany degradation of protein or mRNA and ensure no deterioration in tissuemorphology. We have previously shown that human liver obtained in thisway shows no loss or degradation of individual forms of hepatic P450(20). Tissue blocks for immunohistochemistry were fixed in 10% neutralbuffered formalin for 24 hours and then embedded in wax, while tissuesamples for immunoblotting and mRNA analyses were rapidly frozen inliquid nitrogen and stored at −80° C. prior to use.

[0045] The presence of CYP1B1 protein in tissue samples was investigatedusing immunohistochemistry (21) and sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) combined withimmunoblotting (22). Immunohistochemistry ensures the identification ofspecific types of cells containing CYP1B1 and is an ideal technique forinvestigating the presence of CYP1B1 in tumor cells as tumors arecomposed of a variable proportion of tumor cells and non-tumor cells.

[0046] Immunohistochemistry was used to determine the cellularlocalization and distribution of CYP1B1 and was performed on formalinfixed wax embedded sections using two antibodies which recognize CYP1B1.Sites of immunoreactivity were detected using an alkaline phosphataseanti-alkaline phosphatase (APAAP) technique (21). Samples of tumor andnormal tissue were fixed in 10% neutral buffered formalin for 24 hourand then embedded in wax. Sections were cut on to glass slides and forimmunohistochemistry sections of tumors and normal tissues were dewaxedin xylene, rehydrated in alcohol and then washed sequentially in coldwater and 0.05 M Tris-HCl (pH 7.6) containing 0.15 M sodium chloride(TBS). The sections were then immunostained with the CYP1B1 antibodies.Subsequently, monoclonal mouse anti-rabbit immunoglobulin (1/100, DakoLtd, High Wycombe, Bucks; UK), mouse anti-rabbit immunoglobulin (1/100,Dako) and mouse monoclonal APAAP (1/100, Dako) were sequentially appliedto the tissue sections for 30 minutes each. Between antibodyapplications the sections were washed with TBS to remove unboundantibody. Sites of bound alkaline phosphatase were identified usingbromo-chloro-indolyl phosphate and nitro blue tetrazolium as the enzymesubstrate. After incubating the sections for 30 minutes at roomtemperature, the reaction was stopped by washing the sections in coldtap water. The slides were then air-dried and mounted in glycerinejelly. The sections were examined using bright field light microscopy inorder to establish the presence or absence of immunostaining, and itsdistribution. SDS-PAGE and immunoblotting was followed by enhancedchemihuuinescence (ECL) technique as described below. Immunoblotting wasalso performed with a monoclonal antibody to β-actin (clone no. AC-15,Sigma, Poole, Dorset, UK) to show the presence of a positive controlprotein in tumor and normal samples and indicate that there was noevidence of protein degradation in any of the tissue samples.

[0047] The presence of CYP1B1 in each tumor that showed CYP1B1 byimmunohistochemistry, and the absence of detectable CYP1B1 in normaltissues, was confirmed by Western blot analysis. The proteins subjectedto Western blot analysis were from isolated microsome preparations.

[0048] Microsomes were prepared essentially as described (32). Tissuesamples were thawed in 25 ml 0.01 M Tris-HCl buffer, pH 7.4, containing1.15% KCl before being homogenized in 0.01 M Tris-HCl buffer, pH 7.4,containing 0.25 M sucrose, 15% glycerol, using an Ultra-Turraxhomogenizer (type TP 18/2; Janlke and Kunkel A G, Staufen Breisgau,Germany). After centrifugation at 15,000× g for 20 min, the supernatantwas removed and recentrifuged at 116,000× g for 50 min. The pellet wasresuspended a first time in 0.1 M Tris-HCl buffer, pH 7.4, containing15% glycerol, 1 mM EDTA, and recentrifuged at 116,000× g for 50 min. Thepellet was resuspended a second time in Tris-HCl-glycerol-EDTA buffer.Microsomal protein concentration was determined (34).

[0049] A discontinuous polyacrylamide gel system, as described (33) withmodifications (32), was employed for separation of proteins in themicrosomes. 20 μl of a 1 mg/ml preparation of normal samples, and 40 μlof a 0.5 mg/ml preparation of tumor samples, in 0.125 M Tris-HCl, pH6.8, containing 2.35% (w/v) sodium dodecyl sulfate, 5% (v/v)2-mercaptoethanol, and 0.005% bromophenol blue tracking dye were loadedonto the gel. 10 μl of a 1 mg/ml preparation of human liver microsomeswere used as positive controls. Samples were run on a 10% non-gradientgel at 30 mA.

[0050] Following SDS-PAGE, resolved proteins were blotted onto anitrocellulose membrane (Schleicher & Schuell; Dassel, Germany)overnight as described (35). Nonspecific binding site were blocked withPBS containing 2% (w/v) nonfat milk, 0.05% (v/v) TWEEN 20TM for 30minutes at room temperature, with continuous shaking. This buffer wasalso used for washing stages. The nitrocellulose membrane was thenincubated with CYP1B1-specific antibody (1:1000) for 90 minutes and goatanti-rabbit immunoglobulin horseradish peroxidase conjugate (1:2000Bio-Rad Laboratories, Hemel Hempstead, Herts, UK) for 60 minutes. Themembrane was washed for three successive 15-minute periods and one60-minute period after each incubation to remove unbound antibody. Boundhorseradish peroxidase was then visualized with an EnhancedChemiluminescence (ECL) kit (Amersham International, Aylesbury, Bucks,UK). Detection was carried out as described in the ECL protocol, withthe X-ray film (Hyperfilm-ECL; Amersham) being exposed for 30 seconds.

EXAMPLE 1

[0051] Expression of CYP1B1 in Normal Kidney and Kidney Tumors wasInvestigated.

[0052] Nephrectomy specimens (n=10) excised from primary renal cellcarcinoma were used. Samples or normal kidney were taken at leastseveral centimeters distant from the edge of each tumor, and onlymacroscopically viable tumor was sampled. Normal human livers (n=5) wereobtained from renal transplant donors and stored at −80° C. prior touse.

[0053] Microsomes of normal kidney, kidney tumors and normal liver wereprepared and subjected to the SDS-PAGE and immunoblotting procedures inan enhanced chemiluminescence technique (21, 22) using an anti-CYP1polyclonal antibody. Recognition of human CYP1B1 was demonstrated usinga maltose-binding recombinant CYP1B1 fusion protein expressed in E coli.Expressed CYP1A1 and CYP142 were supplied by Dr C L Crespi, GentestCorp, Ma, USA. The results are shown in FIG. 1: lane 1 human liver, lane2 expressed recombinant CYP1B1 protein, lanes 3, 5, 7, 9, 11 normalkidney samples, lanes 4, 6, 8, 10, 12 corresponding kidney tumors. Thesame amount of microsomal protein (30 μg) was loaded into each lane,thus allowing direct comparison between the kidney and liver samples.

[0054] As shown in FIG. 1, the kidney tumors and expressed CYP1B1 show asingle immunoreactive band at 60 kDa corresponding to the molecularweight of expressed CYP1B1. In normal kidney none of the samples showedan immunoreactive band at 60 kDa. In addition, none of the kidney tumorsor normal kidney samples showed the presence CYP1A1.

[0055] Immunoblotting of liver samples showed an immunoreactive band at54 kDa corresponding to the molecular weight of CYP1A2. The intensity ofthe band at 54 kDa showed liver-to-liver variation, whereas there was noCYP1B1 immunoreactive band at 60 kDa in any of the liver samples.

EXAMPLE 2

[0056] The expression of CYP1B1 was also investigated in breast cancerusing immunoblotting.

[0057] Samples of breast tissue were obtained from patients undergoingsurgery either for primary breast cancer or non-neoplastic breastdisease. Immunoblotting was performed on breast cancers obtained fromsix patients (age range 45-67; three non-smokers, information notavailable for three patients), and histologically all these tumors werecarcinomas of no special type. The tissue samples were frozen in liquidnitrogen and stored at −80° C. prior to analysis.

[0058] SDS-PAGE and immunoblotting were carried out as describedpreviously. CYP1B1 was detected using the anti-CYP1 polyclonal antibodyreferred to above. The results are shown in FIG. 2: lane 1 human liver,lane 2 expressed CYP1B1, lanes 3-8 breast tumors. As can be seen, asingle protein band of molecular weight 60 kDa corresponding to themolecular weight of the expressed CYP1B1 protein was identified. Aspreviously, CYP1B1 was not detectable in the liver sample, but CYP1A2was detected.

EXAMPLE 3

[0059] Immunohistochemistry was used to demonstrate the presence ofCYP1B1 specifically in a variety of normal and tumor tissues. Theresults are shown in Table 1 and in FIG. 3.

[0060] Immunohistological localization of CYP1B1 was investigated intumors and normal tissues from invasive ductal carcinoma of the breast,endometrial adenocarcinoma, transitional cell carcinoma of the bladder,diffuse high grade malignant lymphoma, high grade astrocytoma of thebrain, soft tissue sarcoma (malignant fibrous histiocytoma), normalliver, normal kidney, normal small intestine. The antibody used was the218A anti-CYP1B1 polyclonal antibody described above.

[0061]FIG. 3 shows an immunoblot of CYP1B1 in different types of tumorsand normal tissues. Lane 1 normal colon, lane 2 colon adenocarcinoma,lane 3 normal kidney, lane 4 carcinoma of kidney, lane 5 normal breast,lane 6 breast cancer, lane 7 normal jejunum, lane 8 normal stomach, lane9 normal liver, lane 10 malignant mixed Müllerian tumor, lane 11endometrial adenocarcinoma, lane 12 ovarian carcinoma, lane 13 diffuse Bcell lymphoma, lane 14 transitional cell carcinoma, lane 15 lungcarcinoma, lane 16 positive control (dioxin-induced ACHN kidney tumorcells (panel A only). In panel B, the same series of tissue samples havebeen immunoblotted for β-actin, which is present in all normal and tumorsamples. A Coomassie blue stained polyacrylamide gel of the same seriesof tissue samples displayed no evidence of protein degradation. Theresults demonstrated that this P450 is specifically localized to tumorcells, and that there is no CYP1B1 immunoreactivity in normal tissues.TABLE 1 CYP1B1 Expression in Tumor Tissues and Normal Tissues. NormalTumor no pos./ no pos./ Histopathological Tissue no tested no testeddiagnosis Bladder 0/8  8/8 transitional cell carcinoma Brain 0/12 11/12astrocytoma Breast 0/10 12/12 invasive ductal carcinoma Colon 0/10 11/12adenocarcinoma Connective tissue  0/9 8/9 sarcoma Kidney 0/11 11/11clear cell carcinoma n = 10; transitional cell carcinoma n = 1 Liver 0/8not tested not tested Lung 0/8  7/8 squamous carcinoma Lymph node 0/5 9/9 non-Hodgkin's lymphoma Esophagus 0/8  8/8 squamous carcinoma Ovarynot tested  7/7 Adenocarcinoma Skin 0/6  6/6 squamous carcinoma SmallIntestine  0/5 not tested not tested Stomach 0/10  9/10 AdenocarcinomaTestis 0/8 14/14 malignant germ cell tumors Uterus 0/5  7/7Adenocarcinoma n = 5; Malignant mixed Müllerian tumor n = 2 Total 0/123128/133

EXAMPLE 4

[0062] Experiments were conducted to detect CYP1B1 RNA in various tumorand normal tissues.

[0063] Reverse transcription polymerase chain reaction (RT-PCR)experiments to detect CYP1B1 mRNA were carried out as described in McKayet al (23). RNA was extracted from tissue samples and cDNA wassynthesized from the isolated RNA using oligo (dT). The CYP1B1 primershad the following sequences: Forward 5′-AAC TCT CCA TCA GGT GAG GT-3′(SEQ ID NO:3) (nt 2104-2123); Reverse 5′-TAA GGA AGT ATA CCA GAA GGC-3′(SEQ ID NO:4) (nt 2573-3593) giving a PCR product of 489 bp. β-actin wasused as a positive control to confirm the presence and integrity of mRNAin each sample and the β-actin primers which were bought from Stratagene(Cambridge, UK) had the following sequences: Forward 5′-TGA CGG GGT CACCCA CAC TGT GCC CAT CTA-3′ (SEQ ID NO:5) (nt 1067-1105); Reverse 5′-CTAGAA GCA TTT GCG GTG GAC GAT GGA GGG-3′ (SEQ ID NO:6) (nt 1876-1905). PCRwith 35 cycles of amplification for both CYP1B1 and β-actin wasperformed as described (23). The positive control for CYP1B1 was a 2.78kb CYP1B1 cDNA and the negative control was sterile water in place ofcDNA. After PCR 10 μl of the PCR product was electrophoresed on a 1.5%agarose gel which incorporated 0.007% w/v ethidium bromide andvisualized by UV illumination. The CYP1B1 PCR product was sequenced,after purification, by the direct dideoxy sequencing technique with a T7sequencing kit (Pharmacia, Milton Keynes, UK) used according to themanufacturer 's protocol. To further investigate the relative amount ofCYP1B1 mRNA in normal and tumor tissues, semi-quantitative RT-PCR ofnormal and tumor kidney samples was performed using serial dilution cDNAof (24). β-actin mRNA was used as an internal control (29).

[0064]FIG. 4 shows CYP1B1 and β-actin mRNA in normal (A and B) andcorresponding tumor (C and D) samples which have been detected byRT-PCR. Lane 1 kidney, lane 2 colon, lane 3 skin, lane 4 esophagus, lane5 stomach, lane 6 lymph node, lane 7 breast.

[0065] Analysis of the tumors by RT-PCR showed that all tumor samples inwhich CYP1B1 had been identified contained CYP1B1 mRNA. The PCR productwas of the expected molecular size when analyzed by agarose gelelectrophoresis. Sequencing of the PCR product confirmed identity withCYP1B1.

CONCLUDING REMARKS

[0066] The absence or low level of individual for P450 in most studiesof human cancer (15-18), combined with extrapolation from studies ofrodent hepatic carcinogenesis (25), had led to the general belief thattumor cells do not significantly express P450. However, we have nowshown that CYP1B1 is expressed in a wide variety of malignant tumors ofdifferent histogenetic types and is not present in normal tissues,indicating that this P450 is a tumor specific form of P450. Tumors arecomposed of a variable proportion of tumor cells and non-tumor cells. Toidentify that a protein is tumor specific, it is important todemonstrate that the protein is localized only to tumor cells.Immunohistochemistry allows the direct visualization of tumor cells andhas the spatial resolution to separate tumor cells from non-tumor cells.Furthermore, it is important to show there has been no differentialdegradation of proteins in normal tissue samples compared with tumorsamples and immunoblotting for β-actin (as a positive control protein)showed it to be present in every normal and tumor sample indicatingthere was no protein degradation. In addition, Coomassie blue stainingof the polyacrylamide gels showed no evidence of protein degradation.Moreover, immunohistochemistry of the tumor samples provides its owninternal control as sections of tumor contain non-tumor cells.

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1 6 1 14 PRT Homo sapiens 1 Glu Ser Leu Arg Pro Gly Ala Ala Pro Arg AspMet Met Asp 1 5 10 2 14 PRT Homo sapiens 2 Glu Lys Lys Ala Ala Gly AspSer His Gly Gly Gly Ala Arg 1 5 10 3 20 DNA Artificial SequenceSynthetically generated primer 3 aactctccat caggtgaggt 20 4 21 DNAArtificial Sequence Synthetically generated primer 4 taaggaagtataccagaagg c 21 5 30 DNA Artificial Sequence Synthetically generatedprimer 5 tgacggggtc acccacactg tgcccatcta 30 6 30 DNA ArtificialSequence Synthetically generated primer 6 ctagaagcat ttgcggtggacgatggaggg 30

What is claimed is:
 1. A composition comprising a compound that isselectively metabolized by CYP1B1 to generate a cytotoxic substance thatkills or inhibits the growth of a tumor cell.
 2. The composition ofclaim 1, wherein the compound is non-toxic prior to its metabolism byCYP1B1.
 3. An anti-cancer drug comprising an amount of the compositionof claim 1 effective to kill or inhibit the growth of a tumor cell whenadministered to an individual having cancer.
 4. The anti-cancer drug ofclaim 3, wherein the tumor cell is a tumor cell of the bladder, brain,breast, colon, connective tissue, kidney, lung, lymph node, esophagus,ovary, skin, stomach, testis, or uterus.
 5. A composition comprising acompound that is selectively metabolized by CYP1B1 to generate asubstance that renders a tumor cell susceptible to a cytotoxic agent. 6.An anti-cancer drug comprising an amount of the composition of claim 5effective to render a tumor cell susceptible to a cytotoxic agent whenadministered to an individual having cancer.
 7. The anti-cancer drug ofclaim 6, wherein the tumor cell is a tumor cell of the bladder, brain,breast, colon, connective tissue, kidney, lung, lymph node, esophagus,ovary, skin, stomach, testis, or uterus.
 8. A method of killing orinhibiting the growth of a tumor cell, the method comprising contactinga tumor cell expressing CYP1B1 with the composition of claim 1, whereinthe CYP1B1 expressed by the tumor cell metabolizes the compound togenerate the cytotoxic substance and thereby kill or inhibit the growthof the tumor cell.
 9. The method of claim 8, wherein the tumor cell is atumor cell of the bladder, brain, breast, colon, connective tissue,kidney, lung, lymph node, esophagus, ovary, skin, stomach, testis, oruterus.
 10. A method of rendering a tumor cell susceptible to acytotoxic agent, the method comprising contacting a tumor cellexpressing CYP1B1 with the composition of claim 5, wherein the CYP1B1expressed by the tumor cell metabolizes the compound to generate thesubstance and thereby render the tumor cell susceptible to the cytotoxicagent.
 11. The method of claim 10, further comprising contacting thetumor cell with an amount of the cytotoxic agent effective to kill orinhibit the growth of the tumor cell.
 12. The method of claim 11,wherein the tumor cell is a tumor cell of the bladder, brain, breast,colon, connective tissue, kidney, lung, lymph node, esophagus, ovary,skin, stomach, testis, or uterus.
 13. A method of treating cancer, themethod comprising administering to an individual diagnosed as having atumor an amount of the composition of claim 1 effective to kill orinhibit the growth of a tumor cell in the individual.
 14. The method ofclaim 13, further comprising detecting expression of CYP1B 1 in thetumor prior to the administration of the composition.
 15. The method ofclaim 13, wherein the administration of the composition does not resultin liver toxicity in the individual.
 16. The method of claim 13, whereinthe administration of the composition does not result in systemictoxicity in the individual.
 17. The method of claim 13, wherein thetumor is a tumor of the bladder, brain, breast, colon, connectivetissue, kidney, lung, lymph node, esophagus, ovary, skin, stomach,testis, or uterus.
 18. A method of treating cancer, the methodcomprising: administering to an individual diagnosed as having a tumoran amount of the composition of claim 5 effective to render a tumor cellin the individual susceptible to the cytotoxic agent; and administeringto the individual an amount of the cytotoxic agent effective to kill orinhibit the growth of the tumor cell.
 19. The method of claim 18,further comprising detecting expression of CYP1B1 in the tumor prior tothe administration of the composition.
 20. The method of claim 18,wherein the administration of the composition does not result in livertoxicity in the individual.
 21. The method of claim 18, wherein theadministration of the composition does not result in systemic toxicityin the individual.
 22. The method of claim 18, wherein the tumor is atumor of the bladder, brain, breast, colon, connective tissue, kidney,lung, lymph node, esophagus, ovary, skin, stomach, testis, or uterus.23. A method of preparing an anti-cancer compound, the methodcomprising: selecting a compound that is metabolized by CYP1B1; andpreparing the compound as a non-toxic substance that is metabolized byCYP1B1 to generate a cytotoxic substance that kills or inhibits thegrowth of a tumor cell.
 24. The method of claim 23, wherein the compoundis selected by screening to identify compositions that are metabolizedby CYP1B
 1. 25. The method of claim 23, wherein the compound is selectedby designing compositions that are metabolized by CYP1B1.
 26. A methodof preparing an anti-cancer compound, the method comprising: selecting acompound that is metabolized by CYP1B1; and preparing the compound as anon-toxic substance that is metabolized by CYP1B1 to generate a moietythat renders a tumor cell susceptible to a cytotoxic agent.
 27. Themethod of claim 26, wherein the compound is selected by screening toidentify compositions that are metabolized by CYP1B1.
 28. The method ofclaim 26, wherein the compound is selected by designing compositionsthat are metabolized by CYP1B1.